Device for concentrating and stabilizing conjugated estrogens from mare urine

ABSTRACT

An apparatus for concentrating and stabilizing conjugated estrogens from pregnant mare urine on solid adsorbent supports to obtain a starting material for pharmaceuticals that contain a natural mixture of conjugated estrogens as active ingredient. The apparatus is suitable for decentralized concentration and stabilization of conjugated estrogen mixtures on cartridges in the vicinity of the horses. The adsorber cartridges can be loaded at the site of urine collection so it is unnecessary to transport large volumes of urine to a central processing point. The loading process can take continuously over a period of weeks until the column is saturated. The effluent urine remains at the site where facilities for its disposal are available. Only the loaded cartridge is transported, and transport can occur at longer intervals of up to several weeks. The stability of the conjugated estrogens on the adsorber assures there is no risk of decomposition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 10/112,040, filed Apr. 1,2002, which is a continuation of international patent application no.PCT/EP01/08657, filed Jul. 26, 2001, designating the United States ofAmerica, the entire disclosure of which is incorporated herein byreference. Priority is claimed based on Federal Republic of Germanypatent application no. DE 100 37 3889.5, filed Aug. 1, 2000.

BACKGROUND OF THE INVENTION

The present invention relates to a device for concentrating andstabilizing conjugated estrogens from pregnant mare urine onto solidsupports in order to obtain a suitable starting material for producingpharmaceuticals that contain the natural mixture of these conjugatedestrogens as the active component.

Estrogens are used in the medical field for hormone substitutiontherapy. In particular, estrogen mixtures are used for the treatment andprophylaxis of change-of-life conditions in women after natural orartificial menopause. In this regard, natural mixtures of conjugatedestrogens present in pregnant mares' urine are particularly effectiveand well tolerated.

The dissolved solid content in pregnant mares' urine (hereinafterabbreviated as “PMU”) can naturally fluctuate in a wide range, generally40-90 g total solids per liter. In addition to urea and other typicalcomponents of urine, phenolic substituents in quantities ofapproximately 2-5 wt-% relative to total solids are contained in PMUsolids. These phenolic substituents include cresols anddihydro-3,4-bis[3-hydroxyphenol)methyl]-2(3H)-furanone, known as HPMF.These substances may be present in the free or conjugated form. PMUcontains a natural mixture of estrogens which are very prevalent in theconjugated form, as the sulfuric acid hemiester sodium salt, forexample, (hereinafter abbreviated as “sulfate salt”). The content ofconjugated estrogens (hereinafter abbreviated as “CE”) can be calculatedas estrogen sulfate salt, and ranges between 0.3 and 1 wt-%, relative tototal solids.

Various methods have been described in the prior art for the directpreparation and recovery of conjugated estrogens contained in PMU.Extracts containing conjugated estrogens are usually obtained from PMUby extraction with a polar organic solvent immiscible or sparinglymiscible in water, such as acetic acid ethyl ester, n-butanol, orcyclohexanol, for example. However, numerous problems arise in suchliquid-liquid extractions, such as intense foam formation,sedimentation, emulsification, and strong phase separation. Severalextraction steps are generally required, which results in losses andonly partial recovery of the estrogen. To avoid these disadvantages,therefore, a number of solid phase extraction methods have been proposedin the prior art.

In order to obtain small quantities of urine and plasma liquids foranalytical determination of estrogens by gas chromatography, Heikkinnenet al. (Clin. Chem. 27/7, (1981), 1186-1189) and Shackleton et al.(Clinica Chimica Acta 107 (1980) 231-243) have described solid phaseextraction of estrogens using a cartridge comprising ilanized silica gelcontaining octadecylsilane groups (Sep-Pak® C¹⁸ cartridge, manufacturedby Waters Ass. Inc., Milford, Mass., USA). In this method the estrogenswere eluted from the cartridge with methanol.

In 1968, H. L. Bradlow (see Steroids 11 (1968), 265-272) proposed theuse of Amberlite XAD-2™, a neutral, nonpolar hydrophobic polystyreneresin from Rohm and Haas for the extraction of conjugated estrogens. Thestated adsorption capacity is low. According to Bradlow, optionallydiluted urine was conducted at a low throughput rate through a columncontaining the resin. The estrogens were eluted with methanol orethanol.

Recent patent applications describe methods for obtaining an extractcontaining a natural mixture of conjugated estrogens from mare urine bysolid phase extraction of the mixture of conjugated estrogens frompregnant mare urine on, for example, RP silica gel (International PatentWO 98/08525) or on nonionized, semipolar polymeric adsorbent resins(International Patent WO 98/08526)

In addition to the aforementioned optimization of the direct, completepreparation of pregnant mare urine (PMU) for obtaining natural mixturesof CE, steps preceding the preparation, such as securing protectivestorage for the estrogens and handling the collected urine at thecollection site, are of particular importance for the yield and qualityof the estrogen-containing raw materials and of the natural mixture ofconjugated estrogens isolated therefrom. In this regard, measures tooptimize the transport of the collected estrogen-containing rawmaterials from the collection site to the site of actual preparation andisolation of conjugated estrogens are also desirable.

The complete processing of PMU, for example by solid phase extraction,requires qualified personnel trained in chemical and pharmacologicalmethods in order to observe under well-controlled conditions theexacting requirements for purity and quality of a substance or substancemixture used as a pharmaceutical agent. Collection of the urine (PMU),however, usually takes place in the normal environment of the pregnantmares, that is, at stud farms in rural and often remote locations. As arule, only a simple infrastructure is present here, and the collectionand handling of urine (PMU) is typically carried out by ordinarypersonnel following instructions, so that high standards for handlingthe collected urine cannot be expected.

However, conjugated estrogens in the composition excreted in pregnantmare urine are a complex mixture containing in particular sodium estronesulfate, sodium equilin sulfate, and other CE. It is very important inthe isolation of CE that the PMU be processed as quickly as possible infresh condition. Under extended storage urine rapidly begins todecompose, turning a dark color and giving off the odor of ammonia, withthe CE content declining drastically. The rate of decomposition is afunction of the storage conditions and the purity criteria. For thisreason, the collected urine (PMU), which involves large volumes ofliquid, hitherto has had to be transported daily from the collectionsite to a centralized processing point to assure the most rapidprocessing possible.

Hence, there is a pressing need for suitable methods and apparatus forconcentrating and stabilizing conjugated estrogens contained in pregnantmare urine directly at the collection site, in a manner that is mostprotective of the product and allows the greatest ease in handling, andfor the most efficient transport from the collection site to the site ofprocessing and isolation.

SUMMARY OF THE INVENTION

The object of the present invention, therefore, is to develop a suitabletechnical method for the recovery of a natural mixture of conjugatedestrogens and a suitable device for concentrating and stabilizingconjugated estrogens (CE) from the collected urine of pregnant mares(PMU), while avoiding the aforementioned disadvantages.

In particular, it is an object that the method and the device allowconjugated estrogens from PMU to be concentrated and stabilized at thesite where the urine is collected, in a manner that protects the productand allows ease in handling, and allow the most efficient transport ofthe estrogen-containing raw material thus prepared, from the collectionsite to the site of actual preparation and isolation of the naturalmixture of conjugated estrogens.

Surprisingly, it has been found that conjugated estrogens bound to solidadsorbent supports are very stable and durable in storage over longperiods of time, with no signs of decomposition.

The present invention, therefore, relates to a method for concentratingand stabilizing mixtures of conjugated estrogens from pregnant mareurine in a manner that meets requirements in the field and isdecentralized, that is, particularly close to the stable or pasture,characterized in that

-   -   the natural mixture of conjugated estrogens contained in        pregnant mare urine is concentrated and stabilized on a solid        adsorbent support, and that    -   a predetermined maximum total amount of collected, optionally        previously strained liquid urine is pumped from a supply vessel,        continuously or in discrete portions, at a predetermined flow        rate through an upright cartridge, and the liquid urine runoff        is discarded, whereby    -   the cartridge is packed with a suitable adsorbent, which is        (continuously) surrounded by liquid, for the adsorption of a        predetermined quantity of the mixture of conjugated estrogens        contained in the liquid urine, with    -   the maximum total amount of liquid urine pumped through the        cartridge, which is predetermined as the end point check for the        loading of the adsorbent, being matched to the maximum loading        capacity of the adsorbent for the conjugated estrogens contained        in the liquid urine.

In the context of the invention, the phrase “meets requirements in thefield, means that the method is adapted at the site to the requirementsof the collection practice, and is therefore simple and safe to manageby trained personnel; that is, it is uncomplicated and capable of beingcarried out with the substantial exclusion of possible operator error.

As used herein, the term “decentralized” means that the method can becarried out at a particular site of the urine collection operationwithout specialized chemical or pharmaceutical apparatus, that is,particularly close to the stable and/or pasture.

The term “cartridge” as used here means any type of closed column havinga closeable inlet and outlet, in addition to connection elements such asquick couplings. Thus, within the scope of the present invention theterm “cartridge” also encompasses columns as they are customarily usedin laboratories, pilot plants, and the chemical industry, for example,and which are equipped to be used according to the invention asdiscussed above.

According to the inventive method, a raw or starting material isprepared which serves for the advantageous production of pharmaceuticalscontaining the natural mixture of conjugated estrogens from PMU as anactive component, in which the natural estrogen mixture content of thePMU is essentially completely concentrated on the adsorbent (support)and can be stabilized over a rather long period under the normalenvironmental conditions of the particular collection site. Aftercomplete (maximum) loading, the cartridge can be easily exchanged andreplaced with a cartridge containing an unloaded adsorbent.

The cartridges with loaded adsorbents may be stored at the site forquite a long time at ambient temperature or optionally underrefrigeration down to approximately 4° C., and, for specificrequirements, also frozen down to approximately −20° C., for example,until a certain number of loaded cartridges is present for practicaltransport to the centralized processing point. The transport may takeplace at ambient temperatures or, for storage as described above, undercooling or refrigeration.

The actual processing and isolation of the natural mixture of conjugatedestrogens (CE) can then occur in a conventional manner in thecentralized processing point for the particular adsorbent, for example,by washing and elution of CE from the adsorbent and further processingof the eluate by customary means. For example, the eluate may be furtherconcentrated in a generally known manner to obtain a concentrate whichis substantially or completely free of organic solvent and suitable forfurther pharmaceutical processing.

If desired, a solids mixture that is free of elution agents may beprepared by other suitable drying methods such as spray drying,fluidized bed drying, freeze drying, or vacuum drying. The eluatecontaining the estrogen mixture as well as a concentrate preparedtherefrom or a spray-dried solid product may be incorporated bycustomary methods into solid or liquid pharmaceutical preparations suchas tablets, dragees, capsules, or emulsions.

In addition to the inventive method, the invention also relates to anapparatus that can be used in the method of the invention, taking intoaccount in a useful manner the requirement for simplicity at the site ofuse and assuring that the method is carried out in a manner that is safeand protective of the product. This apparatus according to the inventionis suitable for concentration and stabilization in a manner that meetsrequirements in the field and is decentralized, that is, particularlyclose to the stable or pasture.

The following description contains a general discussion of severaladditional process parameters that are important in carrying out themethod. With regard to specialized construction or apparative designs ofsuitable devices for carrying out the method according to the invention,reference is made to the following detailed description of the apparatusof the invention, which may be used to supplement the generaldescription of the procedural method given below.

In the method according to the invention, the collected PMU may be usedas such, and to this end the daily collected quantity of PMU is firststored in a supply vessel. As an option, it recommended that coarsemechanical contaminants such as straw, hay, or the like be removedbeforehand, for example by using a simple wide-mesh screen while fillingthe supply vessel with the freshly collected PMU. If desired,preservatives, germicides, bactericides, and/or anthelmintic agents maybe added to the collected urine in the supply vessel to reduce thebacterial and viral counts.

The PMU is regularly pumped from the supply vessel to theadsorbent-containing cartridge once or twice a day, for example,depending on the amount of urine present, using a pump such as a hosepump. This cartridge typically has dimensions and weight which permitmanual handling by a person of average strength. With regard to themethod, it is recommended that the internal dimensions of the cartridgebe designed for the receiving adsorption bed, such that the cartridgecan accommodate approximately 30 to 50 liters of adsorbent, and the PMUis preferably required to travel a path distance of preferably 80 to 120cm from the cartridge inlet to the outlet. According to the invention,the conjugated estrogens are adsorbed onto the adsorbent by contact ofPMU with the adsorbent, whereby the liquid urine is conducted, at eitherthe head or base end, into an upright cartridge containing theadsorbent, and during throughput is maintained in contact with theadsorbent for a sufficient time for the estrogen to be adsorbed, untilthe leftover urine finally exits the opposite end of the cartridge. ThePMU is conducted through the cartridge at the head or base end, forexample, to assure that the adsorbent is continuously surrounded byliquid in order to eliminate to the extent possible undesired dryoperation of the cartridge.

The contact title and the flow rate are matched to the particularadsorbent, and for adsorbents suitable for the method (loading velocity)should be in the range of 3 to 10 adsorber bed volumes/hr, preferably 4to 6 adsorber bed volumes/hr. For example, particularly suitable loadedflow velocities lie in the range from 4.5 to 5.5 adsorber bedvolumes/hr. Particular contact times and flow rates for especiallypreferred adsorbents are described in further detail below.

The maximum total quantity of liquid urine that can be conducted throughthe cartridge is a function of the adsorption capacity of the particularadsorbent, and for adsorbents suitable for the method should be in therange of 20 to 60 adsorber bed volumes, preferably 30 to 40 adsorber bedvolumes. The maximum total quantity of liquid urine that can be ledthrough the cartridge for loading, depending on the adsorbent and thesize of the cartridge, generally is in the range of 900 to 2,000 liters.The particular end value of the throughput quantity is strictlyspecified for the operator at the collection site. Thus, a simple flowmeter, such as a water meter, may be used to measure the quantity ofliquid urine pumped through the cartridge. After the maximum loading isreached, the operation is optionally switched to a parallel, secondcartridge and the adsorption process is continued on this cartridge.

When the predetermined end value for the cartridge is reached, thecartridge with the adsorbed CE may be washed if desired with water, forexample, and/or another suitable aqueous wash solution, before theliquid feed is interrupted. The liquid outlet and the liquid inlet arethen closed and the connection, such as a simple quick coupling, at theliquid inlet to the cartridge is detached. The loaded cartridge can thenbe removed and stored at a suitable location until being transported tothe centralized processing point, and a new, unloaded cartridge can beattached to the liquid inlet.

After loading with PMU, for example before replacing a cartridge whichis completely loaded with PMU or before reaching the total loadingcapacity subsequent to any individual partial loading steps, thecartridge may be rinsed, if desired, with water and/or another suitableaqueous wash solution such as a basic wash solution, especially dilutedaqueous sodium hydroxide, for example (such as aqueous 0.5-2N NaOH) toremove leftover liquid urine from the cartridge. The quantity of washsolution is not critical, and is preferably chosen to be sufficient toexpel leftover urine from the cartridge without also washing outappreciable quantities of conjugated estrogens. It has proven useful touse, for example, one to three, preferably approximately two, bedvolumes of wash liquid per bed volume of adsorbent. In this regard, itis useful to conduct the wash water or wash liquid through the cartridgecontaining the adsorbent at a flow rate of 3 to 10, preferably 5 to 7parts by volume wash water per 1 part by volume adsorbent per hour. Theadsorbent loaded with CE in the cartridge may serve as the raw orstarting material for the isolation of pure CE for the production ofpharmaceuticals containing the natural mixture of conjugated estrogens.

In a variant of the invention, after washing with water and/or washsolution, to further stabilize the adsorbed CE the cartridge may undergoa final rinse with a preservative solution, such as a solutioncontaining a preservative agent, a pH-adjusted aqueous solution, and/oran aqueous salt solution, before detaching the cartridge from the deviceand replacing it with a new cartridge. To prevent bacterialcontamination of the column, particularly during storage or transportwithout refrigeration, customary preservatives and also germicides,bactericides, and/or anthelmintic agents, for example, may be used. A pHadjustment of the wash water depends on the particular adsorbent used.Aqueous solutions of inorganic salts in different concentrations may beused as salt solutions, for which examples are given in Table III.1. Theuse of salt solutions is particularly recommended when the cartridgesloaded with CE are to be stored under cooling or in particular, underrefrigeration. Preferred salt solutions are sodium chloride solutionshaving salt concentrations of approximately 10 to 35 wt-%, preferably 25to 33 wt-%.

In a preferred embodiment of the method according to the invention, thecollected urine is preferably freed of viscous and fine-particle solidsbefore being pumped from the supply vessel to the cartridge. It istherefore useful to first conduct the liquid urine from the supplyvessel through one or more prefilters before pumping the liquid urinethrough the cartridge. In this manner, the PMU can be passed through atleast one conventional separation device, for example, a filtrationsystem having at least one deep-bed filter or precoated filter. As aseparation device, a deep-bed filter having a sand bed, for example, ora commercially available filter cartridge may be used, or alsocommercially available precoated filters or plate filters, cartridgefilters, filter bags, or filtration tubes. The filters may be usedindividually or in any desired combination with one another, forexample, connected successively in series or in parallel.

If desired, filtering aids may be added to the liquid urine (PMU) beforefiltration. Suitable filtering aids include those which bind calciumcarbonate and/or mucin, thereby improving the urine filtration. Thefilter may also be optionally connected to a downstream particleseparator. For example, in the case of a sand bed, the filter may beconnected to a downstream sand separator.

The principles and techniques of filtration are known to those skilledin the art. With regard to the object to be achieved by the invention,the conventional principles of clarification come into consideration.The object of clarification is to purify the liquid phase so that thepurity of the filtrate is the significant parameter for achieving theprocess goal. The separation process is basically implemented by theinteraction between the filter material and the suspension. This isimportant in the selection of the filter material for the processmodeling as well as for the process control design of the filterapparatus.

In most cases, the separated solid exerts a controlling influence on thecourse of the process. Thus, with a sufficient portion of solids and inview of the potential of the solids to form bridges in which particlesare supported over the filter material without plugging the filter, thefilter cake which forms and continuously grows assumes the function ofthe filter material. In this case for coated filtration and cakefiltration, the structure of the filter cake determines the course ofthe process, whereas the actual filter material after an initial phasehas only a supporting function.

The site of the solids separation is a further significant feature fordifferentiating filtration processes. In surface filtration, the solidson the surface of the filter material are separated by a sieve effect,that is, as the result of the size ratio of the solid particles to thepores of the filter material. In contrast, in deep-bed filtration, thesolid particles penetrate into the filter material and settle in theinterior of the material.

The adsorbent, with which the cartridge may be filled, can generally beany inorganic or organic adsorbent that has sufficient adsorptioncapacity for the natural mixture of conjugated estrogens contained inthe PMU. Suitable adsorbents for use in the cartridge thus includepolymeric adsorber resins, silica gel, RP silica gel, and/or preferablysemipolar polymeric adsorber resins.

The hydrophobized silica gels which may be used as adsorbents in thecartridge include, for example, generally known reverse phase silicagels (abbreviated as “RP silica gels”), which are chemically modifiedsilica gels bearing hydrophobic functional groups or ligands. SilanizedRP silica gels which contain n-octadecyldimethylsilyloxy,n-octyldimethylsilyloxy, or dimethylhydroxysilyloxy groups ashydrophobic functional groups, for example, are suitable. Silanizedsilica gels having average particle sizes of, for example, 15 to 500 μmare suitable. Silica gels containing dimethylhydroxysilyloxy groupshaving an average particle size ranging from 0.05 to 0.3 mm, such assilida gel 60/dimethylsilane derivative from Merck, have proven to beparticularly useful.

It is advantageous to conduct the liquid urine through the silicagel-containing cartridge at a throughput rate such that the contact timeis sufficient for adsorption of the estrogen. Throughput ratescorresponding to a throughput of 5 to 20 parts by volume PMU per 1 partby volume silica gel/hour are suitable. The adsorption is preferablycarried out at room temperature. It can be useful to control thethroughput rate of liquid urine through the reactor by operating at aslight positive pressure (controlled by the power of the pump).

The quantity of hydrophobized silica gel to be used may vary, dependingon the type of silica gel used and the solids content in the collectedliquid urine. By using PMU which has been prefiltered (i.e., freed ofviscous and solid materials), one part by volume hydrophobized silicagel, for example, can be loaded with up to eighty parts by volumepretreated PMU without noticeable quantities of estrogen beingdetectable in the liquid urine effluent.

The semipolar polymeric adsorbent resins which may be used in thecartridge as adsorbents are preferably porous organic nonionic polymerswhich, in contrast to nonpolar hydrophobic polymeric adsorbent resins,have an intermediate polarity (for example, having a dipole moment ofthe active resin surface in a range of 1.0 to 3.0, particularly 1.5 to2.0, Debye) and a slightly hydrophilic structure, for example,polycarboxylic acid ester resins. It is advantageous to use macroporoussemipolar resins having a preferably macroreticular structure andaverage pore diameters ranging from 50 to 150, preferably 70 to 100 Å,and a specific surface ranging from 300 to 900, preferably 400 to 500m²/g. Macroporous crosslinked aliphatic polycarboxylic acid esterresins, especially crosslinked polyacrylic ester resins such asAmberlite XAD-7® from Rohm and Haas, have proven to be particularlysuitable.

It is useful to conduct the liquid urine through the cartridgecontaining the adsorbent resin with a throughput rate such that thecontact time is sufficient for adsorption of the estrogen. Suitablethroughput rates correspond, for example, to a throughput of 3 to 10,preferably 5 to 7 parts by volume PMU per 1 part by volume adsorberresin/hour. The adsorption is preferably carried out at roomtemperature. The throughput rate of liquid urine through the reactor maybe advantageously controlled by operating at a slight positive pressure(controlled by the power of the pump). The quantity of semipolaradsorber resin to be used can vary depending on the type of adsorberresin employed and the solids content in the collected liquid urine. Byuse of PMU, one part by volume adsorber resin, such as crosslinkedaliphatic polycarboxylic acid ester adsorber resin, for example, may beloaded with up to eighty parts by volume pretreated PMU withoutnoticeable quantities of estrogen beingdetectable in the liquid urineeffluent.

In addition to the aforementioned preferred adsorbents, other types ofadsorber resins or silica gels may also be used. In this regard,nonpolar, semipolar, and even polar adsorber resins are suitableadsorber resins. The quantity of resin that can be pumped through theadsorbent is determined beforehand, based on the particular adsorbentcapacity. Examples of adsorber resins which may be used includecommercially available types such as polymeric Amberlite adsorbentshaving a styrene divinylbenzene backbone chain (for example, XAD-1180,XAD-2, XAD-4, XAD-16), an acrylic ester backbone chain (for example,XAD-7), or highly polar backbone chains containing nitrogen and oxygen(for example, XAD-12). Other adsorber resins are Dowex resin (copolymersof styrene and divinylbenzene), such as Dowex 112, Dowex Optipore, DowexOptipore V 493; Lewatits (crosslinked polystyrenes) such as Lewatit OC1064, Lewatit OC 1066, or Lewatit OC 1163; and polyamine anion exchangeresins such as Dowex resins. Adsorber resins XAD-7, XAD-16 (HP type),XAD 118, and Dowex Optipore, preferably Dowex Optipore V 493, andLewatits OC 1064, OC 1066, and OC 1163 are particularly advantageous.

In addition to the aforementioned inventive method, the inventionfurther relates to an apparatus which may be used in the method of theinvention, and which effectively takes into account the simpleconditions at the site of use and assures that the method may be carriedout in a safe manner that protects the product.

The apparatus according to the invention is characterized by itssuitability for concentrating and stabilizing mixtures of conjugatedestrogens (CE) contained in pregnant mare urine (PMU) onto a solidsupport (adsorbent) in a manner that meets requirements in the field andis decentralized, that is, located near the stable or pasture. Theapparatus comprises:

-   -   an upright cartridge packed with a suitable adsorbent, which is        (continuously) surrounded by liquid, for the adsorption of a        predetermined quantity of the mixture of conjugated estrogens        contained in the liquid urine of pregnant mares, with the        cartridge having either a) a liquid inlet situated at the base        end and a liquid outlet situated at the head end or b) a liquid        inlet situated at the head end and a liquid outlet situated at        the base end, and    -   a pump, a flow meter, and a throughput meter arranged in the        stated sequence upstream of the cartridge and connected to one        another by hose lines.

The apparatus according to the invention is intended to carry out theaforementioned method for concentrating and stabilizing conjugatedestrogens (CE) contained in PMU in a manner that meets requirements inthe field and is decentralized. If desired, the device may also containtwo cartridges connected in parallel which may be operated concurrentlyor alternately.

The cartridge used in the apparatus according to the invention may bedesigned in a broad dimensional range with regard to its height anddiameter. However, in order not to impair handling in the field, thesize and weight of the cartridge should be designed so that it is easilyhandled and carried by hand, for example, during replacement of loadedcartridges with new, unloaded cartridges, by a person of averagestrength. For example, the cartridge should be designed so that theinternal dimensions of the adsorption bed can accommodate 30 to 50liters of adsorbent, and the internal height in particular is in the 80to 120 cm range. The inner diameter of the cartridge generally rangesfrom 10 to 25 cm. Those skilled in the art are able to design precisedimensions of the cartridge to assure optimal flow conditions duringoperation.

Furthermore, the cartridge used in the apparatus according to theinvention preferably is made of robust, durable, and chemicallyresistant materials as customarily used in the production of devices forthe chemical industry, such as impact-resistant laboratory glass,plastic, and/or metal such as sheet steel.

Any pump with a simple and robust construction may be used in the deviceaccording to the invention, such as a monopump, hose pump, or membranepump. For example, a hose pump has proven to be a useful pump for theapparatus of the invention.

Any flow meter with a simple and robust construction may be used in thedevice according to the invention, such as a rotameter, vane air flowmeter, or inductive flow meter. For example, a rotameter with a floathas proven to be a useful flow meter for the apparatus of the invention.

Any throughput meter with a simple and robust construction may be usedin the device according to the invention to measure the quantity ofliquid urine pumped through the cartridge, such as a water meter orinductive flow meter. A water meter, for example, has proven to be auseful flow meter for the apparatus of the invention.

In one useful variant of the apparatus according to the invention, atleast one or more prefilters, such as deep-bed filters or precoatedfilters, are connected in-line between the pump and the flow meter. Thefilter may be a deep-bed filter having a sand bed or a commerciallyavailable filter cartridge or a commercially available precoated filter,plate filter, cartridge filter, filter bag, or filtration tube. Inaddition, a particle separator may optionally be connected downstream ofthe prefilter, such as the example of a sand separator connecteddownstream of a sand bed being used as a prefilter.

In variants of the invention, the filters may be used singly or in anydesired combination with one another, such as connection in seriesand/or in parallel. An advantageous embodiment of these variants ischaracterized in that the prefilter is a module composed of twoprefilters connected in parallel which are individually operated inalternation, and which may be replaced by new prefilters when the deviceis running.

A further advantageous embodiment of this variant is characterized inthat the filter system comprises a module composed of a prefiltrationunit, connected in series, made of prefilters (filter bags, for example)in addition to one or two deep-bed filters and two absolute filters(membrane filters, for example) connected thereto in parallel, theabsolute filters being individually operated in alternation, and whichalso may be replaced by new filters when the device is running.Furthermore, to check proper functioning of the prefilter it may beuseful in the device according to the invention to connect an in-linemanometer between the pump and the prefilter and an in-line manometer inthe region downstream of the prefilter, preferably just behind the flowmeter.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in further detail hereinafter withreference to illustrative preferred embodiments depicted in theaccompanying drawings, in which:

FIG. 1 is a schematic illustration one possible embodiment of anapparatus according to the invention for carrying out the method of theinvention; and

FIG. 2 is a block diagram illustrating the method and apparatus of theinvention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As noted, FIG. 1 shows a schematic apparatus for stabilizing andconcentrating conjugated estrogens from pregnant mare urine according toa preferred embodiment of the invention. FIG. 2 is a schematicrepresentation of the method for a variant of filtering raw urine.

The method according to the invention and the apparatus according to theinvention have a number of advantages, of which the most important willbe briefly stated below. Concentration of conjugaated estrogens, thatis, loading of the adsorption columns, may be carried out directly atthe collection site, thereby eliminating the daily transport of largequantities of liquid urine to a centralized processing point. Loadingmay be performed continuously over a rather long time, up to severalweeks, until the adsorption column is saturated. For example, usingadsorber resin XAD-7, the quantity of adsorbed urine is approximatelythirty-five bed volumes.

The leftover urine freed of conjugated estrogens remains at the site,where suitable devices for disposal are already available. The loadedcolumn, that is, only about 1/35 of the original weight of the liquidurine, is transported. Transport may take place at rather long timeintervals and may take longer, for example, up to a few weeks. Theexcellent stability of the CE on the adsorption column ensures thatthere is no risk of decomposition. There is no treatment of largequantities of urine in a separator and an ultrafiltration facility,since any viscous substances and sediment present are separated from theinitially collected urine by using robust and maintenance-free precoatedfilters or deep-bed filters (for example, sand filters, filtercartridges, precoated filters, plate filters, cartridge filters, filterbags, or filtration tubes) which, if necessary, may be easily replaced.

FIG. 1 schematically illustrates an advantageous device for stabilizingand concentrating conjugated estrogens from pregnant mare urine. Theelements of the apparatus identified by reference numerals have thefollowing meanings: (1) inlet for the liquid urine from the supplyvessel, (2) pump, such as a hose pump, (3) connecting lines, (4)manometers, (5) three-way line, (6) quick couplings, (7) closure valves,(8) filter, (9) flow meter with float (rotameter), (10) throughputmeter, such as a water meter, (11) cartridge or column, (12) adsorberbed, (13) outlet.

FIG. 2 is a schematic representation of a method for filtering rawurine, with the filter system comprising a module composed of aprefiltration unit made of prefilters (filter bags, for example) inaddition to one or two deep-bed filters and two absolute filters(membrane filters, for example) connected thereto in parallel.

The invention is further illustrated by the following non-limitingexamples:

EXAMPLE 1

General operating specification for concentrating and stabilizingconjugated estrogens contained in pregnant mare urine directly at thePMU collection site by adsorption onto an adsorbent contained in acartridge.

The collected PMU is filled into a supply vessel via a funnel fittedwith a sieve. The sieve has a large mesh aperture and serves the solepurpose of separating coarse mechanical contaminants such as straw, hay,or the like.

The method is explained below by example, using an adsorption column (asa cartridge) containing a semipolar adsorber resin. If cartridgescontaining other adsorbents are used, the handling and especially theprocessing of the loaded cartridge may depend on the particular type ofadsorbent. The loading process and final washing of the cartridge at theurine collection site were kept essentially as described under A) and B)or, optionally, minimally adapted to any special conditions at the siteor to the particular adsorbent.

A) Adsorption of Estrogen in PMU Onto an Adsorbent

The adsorption of CE contained in PMU was carried out in an adsorptionunit, as shown by example in the sketch in FIG. 1. A predetermined totalquantity of PMU, relative to the capacity of the cartridge or theadsorption column, was pumped in whole or in part at ambient temperatureand at a predetermined throughput rate through a column or cartridgepacked with an adsorbent, after optionally straining and filteringbeforehand. As the adsorber, for example, a semipolar polyacrylic esteradsorber resin (Amberlite XAD-7 from Rohm and Haas, particle size 0.3 to1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 Å, specificsurface approximately 450 m²/g, dry) swollen in water, or other suitableadsorbents were used. After reaching the predetermined maximum loadingquantity of PMU, the PMU feed to the column or cartridge was stopped andoptionally pumped from the loaded column or cartridge to a free columnor cartridge connected in parallel. The estrogen in the PMU wascompletely adsorbed on the adsorption column thus loaded.

B) Washing of the Loaded Adsorption Column

After the adsorption process had ended, the loaded adsorption column orcartridge was facultatively washed with water or another aqueous washsolution such as a basic wash solution, particularly diluted sodiumhydroxide solution (0.5-2 N NaOH, for example). To this end, the washwater or wash solution was likewise pumped through the column orcartridge at a predetermined throughput rate of, for example,approximately 5.5 bed volumes per hour. The discharged wash water wasdiscarded.

C) Storage and Transport

For transport or intermediate storage of the loaded adsorption column orcartridge, after conclusion of the adsorption process or afterfacultative washing (for example, with water or another aqueous,optionally basic wash solution, particularly sodium hydroxide solution,0.5-2 N NaOH, for example), the column or cartridge was closed off atboth ends, removed from loading apparatus, and stored in a suitablelocation. Storage may occur at ambient temperatures, such as 15 to 30°C., in a standard storage room, or in a refrigerator at temperaturesdown to approximately 4° C. If the liquid in the adsorption column isprotected from freezing, refrigeration can be carried out at lowertemperatures, for example, at deep cooling temperatures ranging toapproximately −20° C. The loaded columns or cartridges were transportedfrom the collection and storage site, in regular intervals of severaldays to a few weeks, to a centralized processing point for chemicalprocessing and isolation of the conjugated estrogens by trainedpersonnel.

D) Desorption of Conjugated Estrogens from the Adsorption Column orCartridge

The loaded columns or cartridges were processed in a customary mannerfor separation of accompanying substances and isolation of conjugatedestrogens appropriate for the particular adsorbent. The processing cantake place as described in International Patent Application WO 98/08526by use of a semipolar adsorber resin of Amberlite such as XAD-7. To thisend, the elution liquid was conducted at a suitable flow rate throughthe column or cartridge preheated to 45° C. The discharged eluate wascollected in fractions. The first fraction was approximately 1 bedvolume, while the remaining fractions were each approximately 0.75 bedvolume. The individual fractions were analyzed by HPLC for estronesulfate salt, cresol, and HPMF content. The first fraction was collectedas long as the eluate was colorless to pale yellow. This fractionconsistently contained only traces of estrogen sulfate salt.

After the first bed volume of eluate had been discharged, the eluateturned an intense dark brown color. Approximately 80 to 90% of the totalamount of conjugated estrogens adsorbed on the column was consistentlycontained in the subsequent 2 to 4 fractions. The remaining fractionscontained only slight amounts of estrogen sulfate salt. This was alsoclearly evident by the decrease in color intensity. After distilling offthe solvent, the residual fractions may optionally be further processedand additional conjugated estrogens isolated.

The main fractions containing the conjugated estrogens each had a hightotal solids (TS) content, determined by HPLC, of estrone sulfate salt,and were sufficiently freed of cresol and HPMF so that these fractions,as is, represented suitable extracts for further pharmaceuticalprocessing.

E) Regeneration of the Adsorber Resin Column

The loaded columns or cartridges were regenerated in a customary mannerappropriate for the particular adsorbent. The regeneration can takeplace as described in International Patent Application WO 98/08526 byuse of a semipolar adsorber resin of Amberlite such as XAD-7. For theregeneration, the column or cartridge was first washed, for example withan ethanol/water mixture containing 50 wt-% ethanol adjusted to pH 12,then with a 10 wt-% aqueous sodium citrate solution, once again with theethanol/water mixture, and finally with distilled water. For theadsorber resin referenced as an example, the entire regeneration wascarried out at a temperature of 45° C., whereby here as well theregeneration and wash solutions were passed through the column orcartridge at either the base end or the head end. The column orcartridge can be loaded and regenerated multiple times, up to 40 times,for example.

EXAMPLE 2 Stability Tests with Loaded Adsorption Columns for theDetermination of Desorbed Conjugated Estrogens

Stability tests were performed with adsorption columns loaded withinitially collected urine to examine the column stability, that is, thestability of the adsorbed conjugated estrogens. The adsorption columnswere loaded in a customary manner, then the adsorber resin was takenfrom the columns and stored at two temperatures for different periods oftime. At the end of the storage time, the adsorbate was eluted andanalyzed for conjugated estrogens and accompanying substances. Theresults were compared to the elution results from control samples, thatis, samples that had not been stored.

A) Test Procedure

The absorption was carried out in a 3 liter column packed in a knownmanner, using semipolar adsorber resin of type Amberlite XAD-7(semipolar polyacrylic ester resin, that is, Amberlite XAD-7 from Rohmand Haas, having a particle size 0.3 to 1.2 mm, a dipole moment of 1.8Debye, an average pore diameter of 80 Å, and a specific surface ofapproximately 450 m²/g, dry). To this end, a quantity of 90 liters (30bed volumes) of previously ultrafiltered mare urine from Poland wascharged on the column at room temperature and a throughput rate of fivebed volumes per hour. The estrogen in the pregnant mare urine wascompletely adsorbed on the semipolar adsorber resin column loaded inthis manner. After the column was loaded with the mare urine, the columnwas subsequently washed with 3 liters of water, and the loaded resin wasdischarged from the column and divided inito 10 samples of 300 ml each.These samples were transferred to smaller columns. One sample wasdiscarded.

The following procedures were then carried out:

-   1. Immediate elution of the control sample, that is, without    storage.-   2. Storage of each of the remaining samples at 4° C. or at 25° C.-   3. Elution of samples 1 (4° C.) and 2 (25° C.) after one week of    storage.-   4. Elution of samples 3 (4° C.) and 4 (25° C.) after two weeks of    storage.-   5. Elution of samples 5 (4° C.) and 6 (25° C.) after five weeks of    storage.-   6. Elution of samples 7 (4° C.) and 8 (25° C.) after eight weeks of    storage.

The elution was carried out in a known manner by first washing theloaded adsorber resin with an aqueous sodium hydroxide solution. Aftereach washing step, the respective wash liquid was analyzed for estrone(as the sulfate salt), equilin, cresol, and HPMF content, using HPLC.

An ethanol/water mixture (30 wt-%, pH 12) made alkaline by the additionof sodium hydroxide was used as the elution liquid, and the resin waseluted at an elution temperature of 45° C. The eluate was collected infractions, and the individual fractions were analyzed for estrone,cresol, and HPMF content, again using HPLC. The first fraction wascollected as long as the eluate was colorless to pale yellow. Thisfraction contained only traces of estrone (estrogen sulfate salt). Afterthe first bed volume of eluate had been discharged, the eluate turned anintense dark brown color. Approximately 80 to 90% of the total amount ofconjugated estrogens adsorbed on the column was consistently containedin the subsequent 2 to 4 fractions. The remaining fractions forregenerating the resin (each at 45° C. in the referenced sequence, with50 wt-% ethanol at pH 12, 10 wt-% sodium citrate, and water) containedonly slight amounts of estrogen sulfate salt. This was also clearlyevident by the decrease in color intensity.

B) Results

The results of the column stability tests are presented for individualsamples in Tables I.0 through I.9 and summarized in Table I.10, and arediscussed in more detail below. The loading of the column or cartridgewith urine (see Table I.0) and elution of the control column (see TableI.1) showed a normal course of adsorption and desorption. However,elevated values of cresol and HPMF were found in the eluate, which couldbe attributed largely to incomplete laminar flow in the column or to thevery high cresol levels (almost 1000 mg/liter) in the initiallycollected urine.

Tables I.2 through I.9 give the results of elution after storage periodsof one, two, five, and eight weeks. The basic objective was to determinethat, as a rule, the adsorbed hormones (conjugated estrogens) werequantitatively desorbed. Occasionally, particularly after only one weekstorage at 25° C., for example, quantitative desorption was determinedonly during regeneration with 50% ethanol. However, this involved onlydesorption of conjugated estrogen residues, and the majority had alreadybeen isolated in the actual desorption. There appeared to be little orno dependency of desorption on the storage time and temperature,provided, for example, that possible non-optimal flow conditions did notplay a greater role in the small columns. However, in all cases theeluate was essentially free of cresol and HPMF. To obtain optimalresults, therefore, attention should be directed to optimal columnthroughput, regardless of the storage time. The columns or cartridgesshould therefore always be immersed in liquid; that is, dry operationshould be avoided.

The results after five weeks' storage presented in Tables I.6 and I.7show a satisfactory course of elution at a temperature of 4° C., whereasat T=25° C., hormone still appears in the regenerate. The HPMF andcresol content in the eluate was very small in both cases.

Surprisingly, excellent results were obtained even after a very longstorage time, as shown in Tables I.8 and I.9 for the eight-week storage.The elution peaks for estrone and equilin were very steep and sharp;that is, the volume of conjugated estrogen eluate was small. The cresoland HPMF values were practically nil.

Table I.10 gives an overview summary of all the tests. The sum of, forexample, the estrone content of the eluate and regenerate was withinapproximately 1055 mg±4% in all cases, in keeping with the expected massbalance.

In summary, it may be concluded that the one- to eight-week storage ofthe loaded XAD-7 resin at temperatures of 4° C. and 25° C. showsdifferences in elution behavior and the height and width of the hormonepeaks, but not in the sum of all hormones eluted, which taken togetherwere in the range of 1055 mg±4%, relative to the estrone content. Itthus follows that the observed qualitative differences are based for themost part only on nonhomogeneous flow conditions in the relatively smallcolumns used, and may be easily avoided by the use of columns orcartridges with dimensions meeting standards in the field.

Table I.0: Column Test for Stability of Column Material

Loading of Column with Native Urine from Poland, Ultrafiltered Adsorber:XAD-7, Volume: 3 Liters

After charging on the column, resin was discharged and subsequentlywashed with 3 liters of water. The resin was divided into equal portionsof 300 ml each and subjected to a column stability test. One resinparcel was eluted immediately, while the others were stored at roomtemperature and under refrigeration. Volume Estrone HPMF Cresol Equilina) Samples liters mg/l mg/l mg/l mg/l Starting solution (afterultrafiltration) Control 30 85.4 67.2 908.0 50.1 solution 1 Control 30117.7 96.9 990.4 70.0 solution 2 Control 30 137.6 105 997.5 80.2solution 3 90 113.567 89.7 965.3 66.8 =30 BV =Average

b) Vol. Time Flow Flow Estrone Equilin HPMF HPMF Cresol Cresol Samplesliters hr l/hr BV/hr mg/l mg/l mg/L mg mg/l mg Run 1 10 0.7 15 5.0 0.00.0 142.6 1426.0 2.7 27.0 Run 2 10 0.7 15 5.0 0.0 0.0 0.0 0.0 2.2 22.0Run 3 10 0.7 15 5.0 0.0 0.0 0.0 0.0 0.8 8.0 Run 4 10 0.7 15 5.0 0.0 0.0148.5 1485.0 1.4 14.0 Run 5 10 0.7 15 5.0 0.0 0.0 150.6 1506.0 5.2 52.0Run 6 10 0.7 15 5.0 0.0 0.0 153.7 1537.0 27.3 273.0 Run 7 10 0.7 15 5.00.0 0.0 153.8 1538.0 81.9 819.0 Run 8 10 0.7 15 5.0 0.0 0.0 155.7 1557.0322.4 3224.0 Run 9 10 0.7 15 5.0 0.0 0.0 154.2 1542.0 770.5 7705.0

TABLE I.1 Column test for stability of column material Elution withoutstorage Adsorber: XAD-7, volume: 300 ml Starting solution: native urinefrom Poland, ultrafiltered Vol. Time Flow Flow Estrone Estrone HPMF HPMFCresol Cresol Equilin Equilin Samples mL min mL/min BV/hr pH TS % mg/Lmg mg/L mg mg/L mg mg/L mg Starting solution: 90 L loaded on a 3-Lcolumn (Table I.1, resin divided into 10 portions of 300 mL each)Starting 7700 113,6 874,7  89,7  690,7   965,3 7432,8  66,8 514,4 content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 25 5,0  9,4102,6 30,8 37,2  11,2 1423,1 426,9 53,6 16,1 Wash 2 300 12 25 5,0 13,1 29,5  8,9 216,0  64,8 5000 1500,0  48,4 14,6 Wash 3 300 12 25 5,0 13,1 12,8  3,8 110,8  33,2 5000 1500,0  30,5  9,2 Wash 4 300 12 25 5,0 13,2 99,8  2,9 45,7  13,7 2000 600,0  9,8  2,9 Wash 5 300 12 25 5,0 13,2 8,4  2,5 54,1  16,2 1023,3 307,0  5.0  1,5 Elution: Ethanol 30%, 45° C.pH 12 Eluate 1 300 12 25 5,0 13,1 3.6 109,2 32,8 585,1  175,5   565,8169,7 56,3 16,9 Eluate 2 300 12 25 5,0 13,0 1,9 2099,2  629,8  0,0  0,0 394,8 118,4 1209,8  362,9  Eluate 3 300 12 25 5,0 12,7 0,6 815,6 244,7 1,0  0,3  119,9  36,0 466,9  140,1  Eluate 4 300 12 25 5,0 12,5 0,2268,8 80,6 0,0  0,0  50,8  15,2 150,2  45,1 Eluate 5 300 12 25 5,0 12,30,1  87,2 26,2 0,0  0,0  21,6  6,5 44,8 13,4 Regeneration: 50% ethanol,45° C., pH 12 Regen 1 600 24 25,0 5,0 12,2  39,1 23,5 0,0  0,0  12,0 7,2 18,8 11.3 Regeneration: 10% Na citrate/water, both at 45° C. Regen2 600 24 25 11,7  2,9 0,0   5,9  1,1 Regen 3 600 24 25 11,3  1,4 0,0  3,8  0,0

TABLE I.2 Column test for stability of column material Elution after 7days storage at 4° C. Adsorber: XAD-7, volume: 300 ml Starting solution:native urine from Poland, ultrafiltered Vol. Flow Flow Estrone EstroneHPMF HPMF Cresol Cresol Eguilin Equilin Samples ML Time mL/min BV/hr pHTS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 L loaded on a3-L column (Table I.1, resin divided into 10 portions of 300 mL each)Starting 7700 113,6 874,7 89,7  690,7  965,3 7432,8  66,8 514,4 content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 25 5,0  9,5 89,6  26,9 88,6  26,6  2604,6  781,4  42,2 12,7 Wash 2 300 12 25 5,013,1  33,7  10,1 343,7  103,1  5396,9  1619,1  58,3 17,5 Wash 3 300 1225 5,0 13,1  9,0  2,7 32,4  9,7 1343,0  402,9   4,7  1,4 Wash 4 300 1225 5,0 13,1  9,1  2,7 0,0 0,0 611,3 183,4   4,7  1,4 Wash 5 300 12 255,0 13,1  9,3  2,8 4,3 1,3 184,0 55,2  4,6  1,4 Elution: Ethanol 30%,45° C., pH 12 Eluate 1 300 12 25 5,0 13,1 3,4 174,2  52,3 4,8 1,4 129,038,7 92,1 27,6 Eluate 2 300 12 25 5,0 13,0 1,7 1362,7  408,8 0,0 0,0167,2 50,2 801,0  240,3  Eluate 3 300 12 25 5,0 12,6 0,6 887,8 266,3 0,00,0  99,5 29,9 512,8  153,8  Eluate 4 300 12 25 5,0 12,4 0,4 433,6 130,10,0 0,0  52,9 15,9 250,0  75,0 Eluate 5 300 12 25 5,0 12,4 0,2 231,0 69,3 0,0 0,0  30,7  9,2 133,4  40,0 Regeneration: 50% ethanol, 45° C.,pH 12 Regen 1 600 24   25,0 5,0 12,4 171,6 103,0 0,0 0,0  27,8 16,7 96,157,7 Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 600 2425 12,1  18,8  11,3 0,0 0,0  0,0  0,0  0,0  0,0 Regen 3 600 24 25 11,2 0,0  0,0 0,0 0,0  0,0  0,0  0,0  0,0[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.3 Column test for stability of column material Elution after 7days storage, 25° C. Adsorber: XAD-7, volume: 300 L Starting solution:native urine from Poland, ultrafiltered Vol. Time Flow Flow EstroneEstrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL min mL/minBV/hr pH TS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 Lloaded on a 3-L column (Table I.1, resin divided into 10 portions of 300mL each) Starting 7700 113,6 874,7 89,7  690,7  965,3 7432,8  66,8514,4  content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 255,0 10,0  98,7  29,6 168,7  50,6  4051,7  1215,5  66,0 19,8 Wash 2 30012 25 5,0 13,0  26,1  7,8 208,3  62,5  4701,1  1410,3  48,4 14,5 Wash 3300 12 25 5,0 13,1  8,0  2,4 55,0  16,5  3122,5  936,8   6,6  2,0 Wash 4300 12 25 5,0 13,1  8,3  2,5 30,6  9,2 1710,5  513,2   5,0  1,5 Wash 5300 12 25 5,0 13,3  8,7  2,6 15,1  4,5 652,9 195,9   5,8  1,7 Elution:Ethanol 30%, 45° C., pH 12 Eluate 1 300 12 25 5,0 13,1 3,4 182,6  54,811,2  3,4 323,0 96,9 96,1 28,8 Eluate 2 300 12 25 5,0 13,0 1,7 904,3271,3 0,0 0,0 216,5 65,0 521,3  156,4  Eluate 3 300 12 25 5,0 12,8 0,8949,0 284,7 0,0 0,0 138,6 41,6 552,1  165,6  Eluate 4 300 12 25 5,0 12,50,5 611,1 183,3 0,0 0,0  82,7 24,8 347,3  104,2  Eluate 5 300 12 25 5,012,4 0,4 343,3 103,0 0,0 0,0  46,9 14,1 192,4  57,7 Regeneration: 50%ethanol, 45° C., pH 12 Regen 1 600 24   25,0 5,0 12,5 191,8 115,1 0,00,0  32,0 19,2 102,7  61,6 Regeneration: 10% Na citrate/water, both at45° C. Regen 2 600 24 25 12,3  19,0  11,4 0,0 0,0  7,9  4,7 10,0  6,0Regen 3 600 24 25 11,3  0,0  0,0 0,0 0,0  0,0  0,0  0,0  0,0[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.4 Column test for stability of column material Elution after 14days storage at 4° C. Adsorber: XAD-7, volume: 300 ml Starting solution:native urine from Poland, ultrafiltered Vol. Time Flow Flow EstroneEstrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL min mL/minBV/hr pH TS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 Lloaded on a 3-L column (Table I.1, resin divided into 10 portions of 300mL each) Starting 7700 113,6  874,7  89,7  690,7  965,3  7432,8   66,8514,4  content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 255,0  9,3 77,3 23,2  42,6  12,8  1255,0  376,8  55,9 16,8  Wash 2 300 1225 5,0 13,1 28,0 8,4 454,6  136,4  6000,0  1800,0   139,3  41,8  Wash 3300 12 25 5,0 13,1  8,8 2,6 30,2  9,1 494,3  148,3   9,4 2,8 Wash 4 30012 25 5,0 13,1  8,2 2,5 0.0 0,0 44,3 13,3   5,3 1,6 Wash 5 300 12 25 5,013,2  8,0 2,4 0.0 0,0 14,5 4,4  4,7 1,4 Elution: Ethanol 30%, 45° C., pH12 Eluate 1 300 12 25 5,0 13,1 3,5 79,8 23,9  5,0 1,5 34,2 10,3  45,613,7  Eluate 2 300 12 25 5,0 12,8 1,8 2528,0  758,4  0,0 0,0  0,0 0,01625,8  487,7  Eluate 3 300 12 25 5,0 12,6 0,4 577,8  173,3  0,0 0,056,8 17,0  335,6  100,7  Eluate 4 300 12 25 5,0 12,6 0,1 131,0  39,3 0,0 0,0 20,9 6,3 81,5 24,5  Eluate 5 300 12 25 5,0 12,6 0,1 38,5 11,6 0,0 0,0  8,8 2,6 23,6 7,1 Regeneration: 50% ethanol, 45° C., pH 12 Regen1 600 24   25,0 5,0 12,6 21,2 12,7  0,0 0,0  9,6 5,8 12,1 7,3Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 600 24 2512,4  4,0 2,4 0,0 0,0  2,4 1,4  2,3 1,4 Regen 3 600 24 25 11,4  0,0 0,00,0 0,0  0,0 0,0  0,0 0,0[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.5 Column test for stability of column material Elution after 14days storage at 25° C. Adsorber: XAD-7, volume: 300 ml Startingsolution: native urine from Poland, ultrafiltered Vol. Time Flow FlowEstrone Estrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL minmL/min BV/hr pH TS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution:90 L loaded on a 3-L column (Table I.1, resin divided into 10 portionsof 300 mL each) Starting 7700 113,6 874,7  89,7  690,7  965,3  7432,8  66,8 514,4  content: Washing: water with NaOH (2%), pH 13 Wash 1 300 1225 5,0  9,3  25,9  7,8 502,5  150,8  6000,0  1800,0   130,0  39,0 Wash 2300 12 25 5,0 13,0  65,5 19,7 130,1  39,0  2188,5  656,6  53,4 16,0 Wash3 300 12 25 5,0 13,1  8,6  2,6 26,8  8,0 707,3  212,2  10,0  3,0 Wash 4300 12 25 5,0 13,1  7,2  2,2 5,2 1,6 76,1 22,8   3,3  1,0 Wash 5 300 1225 5,0 13,1  7,6  2,3 0,0 0,0 32,2 9,7  4,5  1,4 Elution: Ethanol 30%,45° C., pH 12 Eluate 1 300 12 25 5,0 13,1 3,5 170,5 51,2 3,7 1,1 55,816,7  97,3 29,2 Eluate 2 300 12 25 5,0 13,0 1,6 1586,2  475,0  3,7 1,1 0,0 0,0 927,0  278,1  Eluate 3 300 12 25 5,0 12,5 0,6 807,0 242,1    00,0  0,0 0,0 499,3  149,8  Eluate 4 300 12 25 5,0 12,5 0,3 325,4 97,61,2 0,4 41,8 12,5  214,0  64,2 Eluate 5 300 12 25 5,0 12,4 0,2 175,952,8 0,5 0,2 22,7 6,8 106,6  32,0 Regeneration: 50% ethanol, 45° C., pH12 Regen 1 600 24   25,0 5,0 12,4 163,6 98,2 1,1 0,7 25,4 15,2  99,159,5 0.0 Regen 2 600 24 25 11,7  24,5 14,7 0,0 0,0  6,9 4,1 14,7  8,8Regen 3 600 24 25 10,0  0,0  0,0 0,0 0,0  0,0 0,0  0,0  0,8[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.6 Column test for stability of column material Elution after 35days storage, 4° C. Adsorber: XAD-7, volume: 300 ml Starting solution:native urine from Poland, ultrafiltered Vol. Time Flow Flow EstroneEstrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL min mL/minBV/hr pH TS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 Lloaded on a 3-L column (Table I.1, resin divided into 10 portions of 300mL each) Starting 7700 113,6  874,7  89,7  690,7  965,3  7432,8  66,8514,4  content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 255,0  9,2 89,6 26,9  76,4  22,9  1687,2  506,2  64,0 19,2  Wash 2 300 1225 5,0 13,0 48,9 14,7  16,9  5,1 6157,1  1847,1  176,2  52,8  Wash 3 30012 25 5,0 13,1  9,2 2,8 40,5  12,2  899,7  269,9  13,5 4,1 Wash 4 300 1225 5,0 13,1  8,9 2,7 3,0 0,9 96,8 29,0  6,9 2,1 Wash 5 300 12 25 5,013,0  7,6 2,3 0,0 0,0 42,0 12,6  4,3 1,3 Elution: Ethanol 30%, 45° C.,pH 12 Eluate 1 300 12 25 5,0 13,0 3.6 73,3 22,0  5,4 1,6 62,1 18,6 38,911,7  Eluate 2 300 12 25 5,0 12,7 2,0 2437,2  731,2  0,0 0,0 147,1  44,11386,8  416,9  Eluate 3 300 12 25 5,0 12,3 0,5 769,5  230,0  0,0 0,069,0 20,7 438,8  131,6  Eluate 4 300 12 25 5,0 12,6 0,3 223,5  67,1  1,30,4 52,6 15,8 126,6  38,0  Eluate 5 300 12 25 5,0 12,5 0,1 66,7 20,0 0,0 0,0 20,6  6,2 39,8 11,9  Regeneration: 50% ethanol, 45° C., pH 12Regen 1 600 24 25,0 5,0 12,4 15,5 9,3 0,0 0,0 14,3  8,6 15,3 9,2Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 600 24 2512,0  1,4 0,8 0,0 0,0  7,5  4,5  1,1 0,7 Regen 3 600 24 25 10,9  0,0 0,00,0 0,0  0,8  0,5  0,0 0,0[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.7 Column test for stability of column material Elution after 35days storage, 25° C. Adsorber: XAD-7, volume: 300 ml Starting solution:native urine from Poland, ultrafiltered Vol. Time Flow Flow EstroneEstrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL min mL/minBV/hr pH TS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 Lloaded on a 3-L column (Table I.1, resin divided into 10 portions of 300mL each) Starting 7700 113,6 874,7  89,7  690,7  965,3 7432,8  66,8514,4  content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 255,0  9,2  68,8 20,6 91,6  27,5  1595,4  478,6  34,5 10,4 Wash 2 300 1225 5,0 13,0  46,6 14,0 17,0  5,1 6050,3  1815,1  151,6  45,5 Wash 3 30012 25 5,0 13,1  8,8  2,6 3,6 1,1 1168,2  350,5  14,2  4,3 Wash 4 300 1225 5,0 13,1  7,9  2,4 11,7  3,5 351,1 105,3   5,4  1,6 Wash 5 300 12 255,0 13,1  8,0  2,4 0,0 0,0 134,4 40,3  4,7  1,4 Elution: Ethanol 30%,45° C., pH 12 Eluate 1 300 12 25 5,0 13,0 3,5 118,4 35,5 7,5 2,3 101,230,4 64,8 19,4 Eluate 2 300 12 25 5,0 12,9 1,6 1525,9  457,8  0,0 0,0199,2 59,8 811,4  243,4  Eluate 3 300 12 25 5,0 12,6 0,7 987,7 296,3 0,0 0,0 139,1 41,7 512,4  153,7  Eluate 4 300 12 25 5,0 12,4 0,3 479,4143,8  2,2 0,7  71,1 21,3 251,5  75,5 Eluate 5 300 12 25 5,0 12,3 0,3241,0 72,3 0,0 0,0  22,5  6,8 125,7  37,7 Regeneration: 50% ethanol, 45°C., pH 12 Regen 1 600 24   25,0 5,0 12,3 110,9 66,5 1,5 0,9  15,9  9,558,3 35,0 Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 60024 25 11,9  9,6  5,8 0,0 0,0  6,7  4,0  5,6  3,4 Regen 3 600 24 25 10,0 0,0  0,0 0,0 0,0  0,0  0,0  0,0  0,0[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.8 Column test for stability of column material Elution after 56days storage, 4° C. Adsorber: XAD-7, volume: 300 ml Starting solution:native urine from Poland, ultrafiltered Vol. Time Flow Flow EstroneEstrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL min mL/minBV/hr pH TS % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 Lloaded on a 3-L column (Table I.1, resin divided into 10 portions of 300mL each) Starting 7700 113,6  874,7  89,7  690,7  965,3  7432,8   66,8514,4  content: Washing: water with NaOH (2%), pH 13 Wash 1 300 12 255,0  9,2 85,8 25,7 44,5  13,4  1171,3   351,4  44,6 13,4  Wash 2 300 1225 5,0 13,0 74,8 22,4 457,2  137,2  5000,0   1500,0   113,5  34,1  Wash3 300 12 25 5,0 13,1  9,6  2,9 34,1  10,2  702,2  210,7   7,5 2,3 Wash 4300 12 25 5,0 13,2  8,9  2,7 4,3 1,3 63,5  19,1   4.0 1,2 Wash 5 300 1225 5,0 13,1  8,9  2,7 0,0 0,0 23,9  7,2  3.7 1,1 Elution: Ethanol 30%,45° C., pH 12 Eluate 1 300 12 25 5,0 13,1 3,8 53,1 15,9 3,3 1,0 41,7 12,5  27,6 8,3 Eluate 2 300 12 25 5,0 12,8 2,0 2289,1  686,7  5,8 1,70,0 0,0 1288,2  386,5  Eluate 3 300 12 25 5,0 12,4 0,5 869,4  260,8  0,00,0 0,0 0,0 475,9  142,8  Eluate 4 300 12 25 5,0 12,3 0,2 228,2  68,52,4 0,7 0,0 0,0 122,0  36,6  Eluate 5 300 12 25 5,0 12,3 0,1 83,4 25,00,0 0,0 0,0 0,0 45,2 13,6  Regeneration: 50% ethanol, 45° C., pH 12Regen 1 600 24   25,0 5,0 12,2 42,4 25,4 3,0 1,8 0,0 0,0 22,9 13,7 Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 600 24 2511,8  3,1  1,9 0,0 0,0 2,2 1,3  1,5 0,9 Regen 3 600 24 25 10,8  1,1  0,70,9 0,5 0,2 0,1  0,7 0,4[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.9 Column test for stability of column material Elution after 56days storage, 25° C. Adsorber: XAD-7, volume: 300 ml Starting solution:native urine from Poland, ultrafiltered Vol. Time Flow Flow TS EstroneEstrone HPMF HPMF Cresol Cresol Equilin Equilin Samples mL min mL/minBV/hr pH % mg/L mg mg/L mg mg/L mg mg/L mg Starting solution: 90 Lloaded on a 3-L column (Table I.1, resin divided into 10 portions of 300mL each) Starting 7700 113,6 874,7 89,7 690,7 965,3  7432,8 66,8 514,4content: Washing; water with NaOH (2%), pH 13 Wash 1 300 12 25 5,0 9,285,8 25,7 44,5 13,4 1171,3   351,4 44,6 13,4 Wash 2 300 12 25 5,0 13,074,8 22,4 457,2 137,2 5000,0   1500,0 113,5 34,1 Wash 3 300 12 25 5,013,1 9,6 2,9 34,1 10,2 702,2  210,7 7,5 2,3 Wash 4 300 12 25 5,0 13,28,9 2,7 4,3 1,3 63.5  19,1 4,0 1,2 Wash 5 300 12 25 5,0 13,1 8,9 2,7 0,00,0 23,9  7,2 3,7 1,1 Elution: Ethanol 30%, 45° C. pH 12 Eluate 1 300 1225 5,0 13,1 3,8 53,1 15,9 3,3 1,0 41,7  12,5 27,6 8,3 Eluate 2 300 12 255,0 12,8 2,0 2289,1 686,7 5,8 1,7 0,0 0,0 1288,2 386,5 Eluate 3 300 1225 5,0 12,4 0,5 869,4 260,8 0,0 0,0 0,0 0,0 475,9 142,8 Eluate 4 300 1225 5,0 12,3 0,2 228,2 68,5 2,4 0,7 0,0 0,0 122,0 36,6 Eluate 5 300 12 255,0 12,3 0,1 83,4 25,0 0,0 0,0 0,0 0,0 45,2 13,6 Regeneration: 50%ethanol, 45° C. pH 12 Regen 1 600 24   25,0 5,0 12,2 42,4 25,4 3,0 1,81,8 0,0 22,9 13,7 Regeneration: 10% Na citrate/water, both at 45° C.Regen 2 600 24 25 11,8 3,1 1,9 0,0 0,0 2,2 1,3 1,5 0,9 Regen 3 600 24 2510,8 1,1 0,7 0,9 0,5 0,2 0,1 0,7 0,4[see source document for table values; commas in numerical values denotedecimal points.]

TABLE I.10 Summary of the results of the column stability tests SampleSample Sample Sample Sample Sample Sample Sample Loading Cartridge 1 2 34 5 6 7 8 Loading Estrone (mg) 10221 874,7  874,7  874,7 874,7 874,7874,7 874,7  874,7 874,7  Equilin (mg) 6009 514,4  514,4  514,4 514,4514,4 514,4 514,4  514,4 514,4  Storage Temperature — RT 25   4  25  4 25  4  25  4  Duration (days) — — 7  7  14  14   35  35   56  56  Washing Estrone (mg) — 48,9 44,9 45,2  34,6 39,1 42  49,4  49,9 56,4Equilin (mg) — 44,3 39,5 34,4  60,4 64,4  63,2 69,6  44,5 52,1 ElutionEstrone (mg) Resin 1014,1  897,1  926,8  919,6 1006,5  1005,7  1071,2 1087   1056,9   Equilin (mg) discharged, 578,4  512,7  536,7  553,3633,7  529,7 609,2  536,8 587,8  Regeneration divided Estrone (mg) into10 26,0 126,6  114,3  112,9 15,1  72,3 10,1  10,2 28,0 Equilin (mg)portions 13,8 67,6 57,7  68,3  8,7  38,4  9,9  6,0 15,0 Sum of Elution +Regeneration Estrone 1040,1  1023,6  1041,1  1032,5  1021,6  1078,0 1081,3  1097,2  1084,9   Equilin 592.2  580,3  594,4  5601,3  642,4 568,1 619,1  542,8 602,8 

EXAMPLE 3 Stability Tests with Loaded Adsorption Columns for theDetermination of Bacterial Counts

The microbiological quality and harmlessness of natural startingmaterials for the production of pharmaceutical preparations have becomeincreasingly important in recent times. Consequently, analyses wereperformed according to customary methods (pharmacopoeia) to determinebacterial counts in samples after storage of columns for five or eightweeks.

The results of the bacterial count determinations are presented in TableII. The total bacterial counts at a storage temperature of 4° C. werecharacterized as small. At a storage temperature of 25° C., the columnwash water had 1.2 to 4.6*10⁶ organisms, and the eluate had 80 to 170organisms. However, it is noted that the stored column adsorbents werenot washed or treated with preservative beforehand, and thus wereinitially subject to increased risk of bacterial growth. During storagewithout refrigeration, therefore, it is recommended that the loadedcolumns be thoroughly washed at least with water, and advantageouslywith additional stabilizing or preservative wash solutions such assodium chloride solution. TABLE II Stability tests for bacterial countdetermination 5 Weeks Storage 8 Weeks Storage Storage time at ° C. 4° C.25° C. 4° C. 25° C. Column wash water 200 4.6*10⁶ 150 1.2*10⁶ Basic washwater <1 18 3  14 Ethanolic eluate 20 80 3.5 170*Data in CFU/ml (colony-forming units)

EXAMPLE 4 Stability Tests with Refrigerated, Loaded Adsorption Columns

As the result of seasonal or regional conditions, temperatures may fallbelow the freezing point during the storage or transport of loadedcolumns or cartridges, with the undesired result that the aqueouscontents of the columns or cartridges solidify. Corresponding testssupport the conclusion that the freezing process appears to have anegative influence on the elution of rethawed columns or cartridges. Ifthe column cannot be wormed or maintained in a heated environment, or ifstorage takes place at low temperatures, it is recommended that the lastwash be performed with suitable low-freezing mixtures instead of waterto prevent freezing.

A) Test Procedure:

From the numerous possible low-freezing mixtures, of which several aregiven as illustrative examples in Table III.1, a sodium chloridesolution, for example, was chosen for further tests. The addition of 23g NaCl per 100 g water resulted in a freezing point of −21° C. Thecolumn loading proceeded in a conventional manner. After the urine wasloaded, rinsing was performed with 3 bed volumes of water, followed by 2bed volumes of a 33 wt-% NaCl solution. The column was then stored for 9days in a chest freezer at −19° C. TABLE III.1 Examples of liquidaqueous salt mixtures and low-freezing mixtures Achievable g A/100 gTemp. in ° C. Substance A coolant Substance B −3.4 NH₄Cl 23 water −5.3NaNO₃ 43 water −7.8 BaCl₂ 22 ice −10 NaCl 26 water −12 CaCl₂.6H₂O 71water −16 NH₄SCN 57 water −19 (NH₄)₂SO₄ 38 ice −21 NaCl 23 ice −22CaCl₂.6H₂O 45 ice −28 NaBr 39 ice −33 MgCl₂ 22 ice −37 66.1% H₂SO₄ 48snow −40 CaCl₂.6H₂O 55 ice −55 CaCl₂.6H₂O 59 ice

B) Results

Even after 9 days of storage at −19° C., the contents of the columnremained liquid, with no signs of partial solidification. Processing ofthe column, that is, basic washing, elution, and regeneration, proceedednormally. The total hormone content per total solids was approximately22 wt-%. The results are shown in Table III.2.

Negative effects of the high NaCl content on the CE content and theseparation of cresol and HPMF were not detectable. In summary, it may beconcluded that stabilization of urine-loaded columns or cartridges usinga liquid freezing mixture such as an aqueous sodium chloride solution,and storage under refrigeration, for example, down to approximately −20°C., have an advantageous effect, even for periods of 1.5 to 2 weeks. Thecontents of the columns or cartridges remained liquid, and processing ofthe columns or cartridges to isolate conjugated estrogens by washing,elution, and regeneration proceeded normally. Negative effects were notobserved, even at high NaCl concentrations. TABLE III.2 Column test forstability of column material Elution after 9 days storage at −19.1° C.Adsorber: XAD-7, volume: 200 mL Vol. Time Flow Flow TS Cresol CresolEquilin Equilin Estrone Estrone HPMF HPMF Estrone Samples mL min mL/minBV/hr pH % mg/L mg mg/L mg mg/L mg mg/L mg TS % Starting solution: 7 Lloaded on a 3-L column = 35 bed volumes Run 1 1   62 16,1 4,8  61 61 0 00 Run 2 1   61 16,4 4,9  96 96 0 0 0 Run 3 1   62 16,1 4,8 111 111 0 0 0Run 4 1   61 16,4 4,9 111 111 0 0 6 Run 5 1   61 16,4 4,9 275 275 0 0 28Run 6 1   61 16,4 4,9 267 267 0 0 27 Run 7 1   61 16,4 4,9 392 392 0 032 Columns: water and 33% NaOH solution for storage at −19.1° C. w/water 0,6 36 16,7 5,0 646 388 0 0 88 w/ salts 0,4 25 16,0 4,8 465 186 00 5 olution Washing: water with NaOH (2%), pH 13 Wash 1 0,2 12 16,7 5,0 70 14 0 0 0 Wash 2 0,2 11 18,2 5,5 20038  4008 0 70 14 348 Wash 3 0,211 18,2 5,5 4009  802 32 15 3 114 Wash 4 0.2 11 18,2 5,5 287 57 10 2 0 031 Wash 5 0,2 11 18,2 5,5  89 18 9 2 0 0 11 Elulion: Ethanol 30%, 45°C., pH 12 Eluate 1 0,2 12 16,7 5,0 12,1 4,1  81 16 13 3 8 2 5 25 0,02Eluate 2 0,2 12 16,7 5,0 13,3 2,5 266 53 1135 227 2036 407 0 0 8,14Eluate 3 0,2 11 18,2 5,5 12,6 0,5 114 23 493 99 854 171 0 0 17,08 Eluate 4 0,2 11 18,2 5,5 12,4 0,1  62 12 32 6 85 17 4 22 8,50 Eluate 50,2 12 16,7 5,0 12,4 0,1  24 5 8 2 17 3 2 10 1,70 Regeneration: 50%ethanol, 45° C., pH 12 Regen 1 0,4 24 16,7 5,0   14,0 n.b. n.b. n.b.Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 0,4 24 16,75,0   13,0 n.b. n.b. n.b. Regen 3 0,4 24 16,7 5,0 n.b. n.b. n.b. n.b.[see source document for table values; commas in numerical values denotedecimal points.]

EXAMPLE 5 Concentration and Stabilization of Urine in a Field Test

A) General

The objective of a field test is to show that, with sufficient hormonecontent in the urine, satisfactory results can be obtained not only inthe laboratory, but also under production conditions. A test wasconducted under the same conditions that would occur during a samplingsession at any given location.

B) Test Procedure

Fresh urine of sufficient quality was collected at a stud farm. Withthree horses that produced 15 to 20 liters of urine per day, a total ofapproximately 150 to 200 liters of urine was obtained in 10 days.Therefore, a 5 liter adsorption column was chosen for adsorption. Urinecollection was started on a weekend, and the daily loading with urine,excluding weekends, was carried out according to the following program:Monday: Charging of urine from Saturday, Sunday, and Monday Tuesdaythrough Friday: Charging of urine from each day Monday Charging of urinefrom Saturday, Sunday, and Monday Tuesday Elution

The urine was charged directly on the column at a loading rate of about4.5 bed volumes per hour, with only a single cotton plug having aseparation limit of 20 μm serving as a prefilter. In order to make theconditions more severe, no rinsing with water was performed between theindividual charges. In pretests it was possible to deliver up to 450liters of urine through the cotton plug with no pressure loss. In thisfield test, filtration proved to be difficult as a result of anunexpectedly high amount of calcium carbonate sediment and/or mucins.

After each 40 to 50 liters, a blockage appeared, along with a pressurerise of approximately 4 bar, which required the filter to be replaced.Depending on the calcium carbonate and/or mucin content, the optionaladdition of filtration aids is recommended.

Although the plug allowed sediment particles >20 μm in size to passthrough, augmentation of the adsorption column was not indicated.

C) Results

Hormone Content

The results of this field test are presented in Table IV.

The estrone and equilin contents of 123 mg/liter and 74 mg/liter,respectively, in the raw urine were surprisingly high, corresponding tourine of average to good quality. However, the cresol values, at 531mg/liter, were unexpectedly high in fresh urine, which hitherto had notbeen observed. The diet of the animals plays a role here and is a factorwhich should be considered. Although fundamentally, a high cresolcontent, for example, can be overcome, in individual cases it may beproblematic. Therefore, it may be necessary to prolong the alkalinewashing process during processing to minimize the cresol values in theethanolic eluate. Otherwise, the adsorption and desorption proceedednormally. The eluate showed a pronounced content of estrone and equilin.Eluates 2 and 3 contained more than 95% estrone/equilin. The totalhormone content relative to the total solids content was approximately30 wt-%, thus meeting expectations for the test and achieving superiorconcentration to a hormone/total solids content greater than 15 wt-%under severe test conditions. TABLE IV.1 Field test for stabilizing andconcentrating CE Test days: 1-10, column loading (on 7 working days)Adsorber: XAD-7, volume: 5 liters Starting solution: native urine,untreated The column was traversed from bottom to top. Vol. Time FlowFlow TS Estrone Cresol Cresol Equilin Equilin HPMF Estrone/ Samples Lmin mL/min BV/hr pH % Estrone mg mg/L mg Mg/L mg mg/L TS % Startingsolution: 140 L loaded on a 5-L column = 28 bed volumes (total quantityfrom 7 individual charges) Starting 140 9,0 6 112,5    531,1  37,4  42,5content Loading: water with NaOH (2%), pH 13 Run 1 10 26 384.6 4.6 0   0,0 0 0 0 Run 2 10 27 370.4 4.4 0 249 2490 0 0 Run 3 10 27 370.4 4.40 245 2450 0 0 Run 4 10 28 357.1 4.3 0 408 4080 0 0 Run 5 10 27 370.44.4 0 358 3580 0 0 Run 6 10 23 434.8 5.2 0 537 5370 0 0 Run 7 10 29344.8 4.1 0 901 9010 0 0 Run 8 10 29 344.8 4.1 8.7 0 836 8360 0 0 Run 910 29 344.8 4.1 0 581 5810 0 0 Run 10 10 24 416.7 5.0 0 605 6050 0 0 Run11 10 25 400.0 4.8 0 621 6210 0 0 Run 12 10 27 370.4 4.4 0 509 5090 0 0Run 13 10 27 370.4 4.4 0 668 6680 0 0 Run 14 10 24 416.7 5.0 0 655 65500 0[see source document for table values; commas in numerical values denotedecimal points.]

TABLE IV.2 Field test for stabilizing and concentrating CE Processing ofcolumn on Test Day 11 Adsorber: XAD-7, volume: 5 liters Startingsolution: native urine, untreated The column was traversed from bottomto top. Vol. Time Flow Flow Estrone Estrone Cresol Cresol EquilinEquilin HPMF Estrone/ Samples L min mL/min BV/hr pH TS % mg/L mg mg/L mgmg/L mg mg/L TS % Starting 140  9,0 6   112,5  531,1   37,4  42,5content: Washing: water with NaOH (2%), pH 13 Wash 1 5 15 333,3 4,0 01104   5520 0 40  Wash 2 5 15 333,3 4,0 0 4400   22000 0 252  Wash 3 514 357,1 4,3 0 2355   11775 0 60  Wash 4 5 14 357,1 4,3 0 40  200 0 0Wash 5 5 14 357,1 4,3 0 7 35 0 0 Elution: Ethanol 30%, 45° C., pH 12Eluate 1 5  8 625,0 7,5 3,8 23  115 12  60 9 45 0 0,1 Eluate 2 5 10500,0 6,0 2,0 2797   13985 201  1005 1211   6055 0 14,0 Eluate 3 5 12416,7 5,0 0,3 481  2405 0 0 193  965 0 16,0 Eluate 4 5 13 384,6 4,6 0,014  70 0 0 7 35 0 Eluate 5 5 13 384,6 4,6 0,0 0 0 0 0 0 0 0Regeneration: 50% ethanol 45° C., pH 12 Regen 1 10   0,4 0,4 0,0 1  0 —0 0 0 Regeneration: 10% Na citrate/water, both at 45° C. Regen 2 10  0,4 0,4 0,0 11,3 0 0 0 0 Regen 3 10   0,4 0,4 0,0 10,3 0 0 0 0 Regen 42 BV water rinsed from bottom to top[see source document for table values; commas in numerical values denotedecimal points.]Bacterial Counts

In addition to urine adsorption, possible growth of bacteria on thecolumn was investigated. Under the following conditions,

-   1. Charging of contaminated raw urine (the cotton plug filter did    not separate any bacteria),-   2. No rinsing after daily charging,-   3. Test carried out a room temperature, and-   4. Test duration of 10 days,

the bacterial density on the column and subsequent growth thereon werenot precluded. Determination of the total bacterial count at differentadsorption steps gave the following results: Sample Total bacterialcount/ml Raw urine 1.1 * 10⁸ Column wash water after 10 days 1.1 * 10⁸Basic wash water  <1 Main eluate 1.5D) Summary of Results

Fresh urine was charged on a 5 liter adsorption column over a testperiod of 10 days. The entire volume of urine was 140 liters, with anaverage estrone and equilin content of 123 mg/liter and 74 mg/liter,respectively.

Column rinsing with water after the daily urine adsorption wasintentionally omitted. Both of the very pronounced main eluatescontained approximately 93% estrone and equilin. The total hormonecontent relative to total solids was approximately 30 wt-%. A furthersurprising result related to possible bacterial contamination of thecolumn. In spite of charging highly contaminated raw urine, bacterialgrowth on the column itself was not detectable after 10 days. With 1.5organisms/ml, the eluate obtained was practically bacteria-free. Hence,under severe conditions it was proven on a large scale which, withsufficient hormone in the raw urine, stabilization and concentration toa hormone/total solids content of greater than 15% and of good qualitycan be performed using an adsorption column.

EXAMPLE 6 Filtration Tests Using Filter Cartridges

Filtration tests using filter cartridges were carried out in a furtherfield test, the results of which are summarized below.

Related Material: Fresh Urine from a German Stud Farm.

The urine of pregnant mares which had been collected in drums andoccasionally agitated was pumped from the drums using a hose pump,through a 5μ bag (prefilter), and into a container. Sediment of about0.5 wt-% remained in the container. FIG. 2 shows a schematicrepresentation of the method for filtering raw urine.

1st Test: Bag 5μ Filter cartridges 5μ, 3μ, 1μ connected in seriesMembrane filter 2 × 1.2μ connected in parallel Column loading 95 litersof filtrate Sediment filtrate 0.000%

2nd Test: Bag 5μ Filter cartridges 5μ, 3μ, 1μ connected in seriesMembrane filter 2 × 3μ connected in parallel Column loading 142 litersof filtrate Sediment filtrate 0.001%

3rd Test: Bag 5μ Filter cartridges 3μ, 1μ connected in series Membranefilter 2 × 1μ connected in parallel Column loading 160 liters filtrateSediment filtrate 0.000%

In each test, one sample was taken downstream of the membrane filter.After every 100 liters a sample was taken from the outlet, downstream ofthe column. The total column loading was 397 liters of filtrate. Thetotal solids (TS) content after processing was 5.9 wt-%.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the described embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.

1. An apparatus for concentrating and stabilizing mixtures of conjugatedestrogens from pregnant mare urine on a solid adsorbent support,comprising: an upright cartridge packed with an adsorbent, which issurrounded by liquid pregnant mare urine, for the adsorption of apredetermined quantity of a mixture of conjugated estrogens contained inthe pregnant mare urine; said upright cartridge having either a) aliquid inlet situated at a base end and a liquid outlet situated at atop end, or b) a liquid inlet situated at the top end and a liquidoutlet situated at the base end, and a pump, a flow meter, and athroughput meter arranged in sequence with said upright cartridge andconnected to one another by hose lines.
 2. An apparatus according toclaim 1, wherein at least one prefilter is connected in-line between thepump and the flow meter.
 3. An apparatus according to claim 2, whereinsaid at least one prefilter comprises a deep-bed filter or a precoatedfilter.
 4. An apparatus according to claim 2, wherein the prefilter is amodule composed of two prefilters connected in parallel which areindividually operated in alternation, and which may be replaced by newprefilters during operation of the apparatus.
 5. An apparatus accordingto claim 2, wherein a first pressure meter is connected in line betweenthe pump and the prefilter, and a second pressure meter is connectedin-line downstream of the prefilter for monitoring functioning of theprefilter.
 6. An apparatus according to claim 1, wherein the cartridgedefines a chamber for the adsorption bed with an internal height rangingapproximately from 80 to 120 cm and an inner diameter rangingapproximately from 10 to 25 cm.
 7. An apparatus according to claim 1,wherein the cartridge is made of impact-resistant laboratory glass,plastic, or metal.
 8. An apparatus according to claim 1, wherein thepump is a monopump, a hose pump, or a membrane pump.
 9. An apparatusaccording to claim 1, wherein the flow meter is a rotameter with float,a vane air flow meter, or an inductive flow meter.
 10. An apparatusaccording to claim 1, wherein the throughput meter which measures thequantity of liquid urine pumped through the cartridge is a water meteror an inductive throughput meter.